Advancing the Understanding of the Mo-CBP2 Protein: Unraveling Its Antifungal and Antibiofilm Properties Against Candida Species.

Abstract

Candida spp. are opportunistic fungi capable of forming biofilms, a key factor contributing to their resistance to conventional antifungals. This highlights the need for novel compounds with distinct mechanisms of action to combat fungal infections. This study aimed to evaluate the antifungal activity of Mo-CBP₂, a chitin-binding lectin from Moringa oleifera seeds, against Candida albicans and Candida tropicalis, with a specific focus on its effects on planktonic cells and biofilms, and to investigate its mechanism of action. Mo-CBP₂ was purified via affinity and ion-exchange chromatography. Antifungal activity was assessed using microdilution, CFU counts, and MTT assays for planktonic cells, and crystal violet staining for biomass quantification in both early-stage and mature biofilms. Mechanistic studies included aggregation assays, sorbitol protection tests, and ergosterol quantification. Against planktonic cells, Mo-CBP₂ exhibited potent antifungal activity, with MIC₅₀ values ranging from 20-45 μM, reducing metabolic activity and CFUs by up to 90%. Additionally, it promoted aggregation of fungal cells, indicating interaction with cell wall components, and showed a fungistatic profile. Regarding biofilms, Mo-CBP₂ significantly inhibited biomass formation in both initial adhesion and mature stages (CIB₅₀ = 20 μM), with greater efficacy than nystatin. Mechanistic assays revealed that its antibiofilm effect is independent of ergosterol biosynthesis or cell wall synthesis pathways, as no alterations were observed in the presence of sorbitol or in sterol content. Mo-CBP₂ presents strong antifungal and antibiofilm activities against Candida spp., acting through a novel, non-conventional mechanism.